Metal cluster containing nucleotides and nucleic acids, and intermediates therefor

ABSTRACT

Nucleotides including a sugar moiety, a pyrimidine or purine base and a terminal thiol group at a side chain covalently linked to pyrimidine or purine base of the nucleotide, and optionally further including a metal cluster covalently linked through the terminal thiol group at said side chain to the pyrimidine or purine base of the nucleotide, and nucleic acids incorporating same.

FIELD OF THE INVENTION

The present invention relates to nucleotides comprising a terminal thiolgroup, to such nucleotides further comprising a metal cluster,preferably a gold cluster, covalently attached through said terminalthiol group, and to nucleic acids comprising at least one of thedescribed nucleotides.

BACKGROUND OF THE INVENTION

Protein-ribonucleic acid (RNA) interactions play a key role in manyfundamental life processes In living organisms, these polymers are oftenfound complexed together in extremely large assemblies whose molecularmass may reach several millions of daltons. In the pathway of geneexpression one finds transcribing complexes, containing RNA polymerasein action on a DNA template, with associated nascent RNA. Concurrently,the resulting precursor messenger RNA (pre-mRNA) becomes associated witha multitude of proteins and additional small RNA molecules into a largeribonucleoprotein (RNP) complex, the spliceosome, where it is processedto mature mRNA. Protein synthesis then takes place in the cytoplasm on athird class of particles—the ribosomes. In addition, a number of largeprotein complexes require mononucleotides (e.g., ATP, GTP) for theirassembly and/or catalytic activity.

For such inherently polymorphic assemblies, visualization bytransmission electron microscopy (TEM) provides structural informationat a resolution that is difficult to obtain in any other way (Chiu andSchmid, 1997; Griffith et al., 1997). Yet, localization and tracing byelectron microscopy of RNA or ribonucleotides within such largebiological assemblies, are not yet a straightforward undertaking. Evenwhen crystals amenable to X-ray crystallography analysis can beobtained, as is the case for ribosomes, there is still a demand forheavy atom derivatives to phase the diffraction data (Weinstein et al,1992). Covalent derivatization of RNA with heavy atoms should enablevisualization of RNA within RNP complexes by EM and ensure theintroduction of electron-dense centers at distinct locations withincrystallized RNA molecules and RNP complexes.

Visualization of nucleic acid molecules by TEM cannot be directlyachieved because of the low-density weakly scattering atoms theycontain. Nevertheless, methods such as electron spectroscopic imaging(e.g., Bazett-Jones, 1992), tungsten shadow casting (e.g., Wang et al.,1994), atomic force microscopy (AFM; e.g. Hansma et al., 1996; Smith etal., 1997), and scanning tunneling microscopy (e.g., Guckenberger etal., 1994) have been used to visualize naked RNA and DNA molecules. Morerecently, a positive staining protocol that allows visualization ofnucleic acids (Dubochet et al., 1971) was used to visualize RNA strandsemanating from supraspliceosome particles (Muller et al., 1998), yet RNAlocated within the particles was not visible. Tagging suchmacromolecules with clusters of heavy atoms should facilitate theirvisualization by conventional TEM. The present most popular methodemploys colloidal gold noncovalently attached to specific antibodies,protein A or other macromolecular probes. For example, attempts weremade to visualize spliceosomes by dark-field scanning transmissionelectron microscopy (STEM) after tagging with biotinylatedoligonucleotides complementary to the pre-mRNA that had been conjugatedto a streptavidin-colloidal gold complex (Sibbald et al., 1993).

The use of probes with covalently conjugated gold compounds provides anumber of advantages over colloidal gold. These include betterstability, size uniformity, and complete absence of aggregation, all ofwhich result in better sensitivity and resolution. A number of goldclusters containing a core of 11 gold atoms surrounded by a hydrophilicorganic shell of aryl-phosphines have been described (Safer et al.,1986). These undecagold compounds have the general formula Au₁₁L₆L′X₃,where L is tris(4N-methylcarboxamidophenyl)phosphine, and L′ is asimilar ligand in which the methylcarboxamido group on one of thebenzene rings is replaced by an activatable side chain such as anω-amino alkyl group. Activation of this compound with a maleimido groupyields a gold cluster that can be conveniently coupled to free thiolgroups of proteins (Safer et al., 1986; Wenzel and Baumeister, 1995). Aninteresting example is the specific labeling with undecagold of theribosomal protein BL11 within the 50S ribosomal subunit of Bacillusstearothermophilus for its subsequent use as a heavy atom derivative forcrystallographic studies (Weinstein et al., 1989, 1992). The sameauthors also labeled tRNA^(phe) of the same organism by taking advantageof the modified nucleoside 3-(3-amino-3-carboxypropyl) uridine atposition 47. The exposed primary amine of this base was reacted with2-iminothiolane to extend the aliphatic chain and introduce a primarythiol group, which was then coupled to maleimido undecagold (Weinsteinet al., 1992).

The diameter of the undecagold cluster is 0.82 nm. It can thus bevisualized by high-resolution STEM, but not readily by conventional TEMunless the signal is enhanced by silver enhancement (Burry et al.,1992). Visualization by conventional TEM can be improved by using alarger, 1.4 run. gold cluster (Hainfeld and Furuya, 1992). The structureof this reagent, now commercially available from Nanoprobes (StonyBrook, N.Y.) under the trademark “NANOGOLD”. has not yet been reported.It has nevertheless been used successfully to label proteins (Boisset etal., 1992; Hainfeld and Furuya, 1992) as well as the 5′ or 3′ ends ofDNA oligonucleotides (Alivisatos et al., 1996).

The present invention teaches a general systematic strategy forincorporating gold clusters into nucleotides and nucleic acid molecules.

SUMMARY OF THE INVENTION

The present invention provides, in one aspect, a nucleotide comprising aterminal thiol group at a side chain covalently linked to the pyrimidineor purine base of the nucleotide.

In one embodiment, the sugar moiety of the nucleotide of the inventionis ribose; in another embodiment, the sugar moiety is deoxyribose,dideoxyribose or any other ribose analog. The nucleotide of theinvention may be a monophosphate, a diphosphate, a 3′,5′-bisphosphate ora 5′-triphosphate.

The side chain of the nucleotide of the invention carrying the terminalthiol group may be saturated or unsaturated and has 2-20, preferably2-15, most preferably 2-10, carbon atoms, optionally interrupted byheteroatoms selected from O, S or N and/or substituted by groups such as═O, ═NH and/or 1-3 alkyl groups.

In another aspect, the invention relates to a nucleotide having a metalcluster covalently linked through a terminal thiol group of a side chaincovalently linked to the pyrimidine or purine base of the nucleotide Themetal may be Ag, Au, Hg, Pt, Mo or W, but is preferably a gold clustersuch as colloidal gold.

In a further aspect, the invention relates to a nucleic acid comprisingat least one nucleotide of the invention comprising a free terminalthiol group or a metal cluster covalently linked through a terminalthiol group. The nucleic acid may be a RNA or a DNA molecule.

In one embodiment, the nucleic acid molecule is covalently tagged with ametal cluster. The metal may be Ag, Au, Hg, Pt, Mo or W, but ispreferably a gold cluster such as colloidal gold.

The metal-tagged nucleic acids of the invention are useful as probes formacromolecular assemblies such as protein-RNA complexes.

The invention provides general methodologies for the covalent attachmentof gold-clusters to DNA and RNA (nucleic acids) at random locations aswell as at specific locations.

The general strategy for the attachment of gold-clusters at randomlocations in the nucleic acid molecule is depicted in Scheme I andinvolves the following steps:

(i) preparation of precursor deoxyribonucleoside triphosphates (NTPs)and ribonucleoside triphosphates (rNTPs) whose heterocyclic ringcontains substituents with a terminal thiol group (NTP-SH and rNTP-SH.respectively);

(ii) incorporation of these precursor molecules in DNA or RNA inreactions catalyzed by DNA polymerase or RNA polymerase, respectively;and

(iii) attachment of gold-clusters to the free thiol groups, either byreacting with a commercially available maleimido derivative of thecluster, or by reacting with colloidal gold of pre-determined size.

The strategy for the attachment of a gold cluster to a specific locationin the nucleic acid molecule is depicted in Scheme 2 and involves thefollowing steps:

(i) preparation of 3′,5′ deoxyribonucleoside diphosphates (p[dN]p) and3′, 5′ ribonucleoside diphosphates (pNp) whose heterocyclic ringcontains substituents with a terminal thiol group (p[dN]p-SH and pNp-SH,respectively);

(ii) synthesis of the 5′ half of the nucleic acid whose 3′ endnucleotide is the one that precedes the nucleotide to which a goldcluster should be attached;

(iii) addition of p[dN]p-SH or pNp-SH to the nucleic acid made in (ii)by using the DNA ligase or RNA ligase, respectively; and

(iv) dephosphorylation of the 3′ end of the nucleic acid made in (iii),and ligating it to the 3′ half of the nucleic acid.

Ribonucleic acids (RNAs) play a key role in many fundamental lifeprocesses. These polymers are often found complexed with proteins inextremely large particles whose molecular mass may reach severalmillions of daltons (e.g., ribosomes, spliceosomes, and viruses).Structural studies of such RNA-protein complexes should help elucidatetheir mode of action. For the structural analyses of many macromolecularassemblies, electron microscopy (EM) has served an instrumental role.However, localization by EM of RNA within biological complexes is notyet a straightforward undertaking. Here we describe a methodology forthe covalent tagging of RNA molecules with gold clusters, therebyenabling their direct visualization by microscopical methods. Thestrategy involves transcription in vitro of RNAs that carry free thiolgroups, using ribonucleoside triphosphate analogs containing asubstituent with a terminal thiol group on their heterocyclic ring. Thissynthesis is followed by coupling of gold clusters to the thiolatedtranscript through a maleimido group. Visualization of such gold-taggedRNAs by transmission electron microscopy showed spots of gold clusters,with a diameter of 1-2 nm, arranged at nearly regular distances on animaginary curve that corresponds to the RNA chain. This assignment wascorroborated by atomic force microscopy that exhibited images of RNAchains in which knob-like structures, whose height corresponds to thediameter of the gold clusters, were clearly seen. This inventiondemonstrates the potential use of nucleic acids that are covalentlylabeled with gold clusters for the structural characterization ofprotein-RNA complexes and in microelectronic devices.

BRIEF DESCRIPTION OF THE DRAWINGS

The file of this patent contains at least one drawings executed in colorphotograph. Copies of this patent with color photograph(s) will beprovided by the Patent and Trademark Office upon request and payment ofnecessary fee.

FIG. 1 A synthetic route for the preparation of gold-tagged RNAs. Anucleoside triphosphate (NTP) analog containing a substituent with aterminal thiol group on its heterocyclic ring (NTP-SH) is incorporatedinto RNA in a standard run-off transcription reaction, driven by RNApolymerase and using an appropriately cut plasmid as a template fortranscription. The thiolated RNA molecules thus obtained aresubsequently coupled through their thiol groups to a gold clustercontaining a maleimido functional group to yield the gold-tagged RNA.

FIG. 2 Gel electrophoresis of gold-tagged RNA. β-globin pre-mRNA wastranscribed in the presence of UTP-SH (10% of total input UTP).Increasing amounts (5-15 ng) of the gold-tagged β-globin pre-mRNA (lanes1-3) along with unmodified β-globin pre-mRNA (lane 4) were subjected toelectrophoresis on a 2%-agarose gel and transferred to a nitrocellulosemembrane, and gold-containing bands were revealed by silver enhancement.Response to this treatment, in a quantity dependent manner, is seen onlyin lanes where gold-RNA is present. The unmodified β-globin pre-mRNA of497 bases (lane 4) is not seen. The origin and the migration of sizemarkers (in number of bases) are indicated on the left.

FIG. 3 Transmission electron microscopy (TEM) images of unstainedβ-globin pre-mRNA transcripts. RNA was transcribed in vitro, treatedwith monomaleimido NANOGOLD, and visualized by bright-field TEM. (a)Unmodified RNA; (b) RNA transcribed in the presence of ATP-SH (10% oftotal input ATP); (c) RNA transcribed in the presence of UTP-SH (50% oftotal input UTP). Arrows indicate the RNA termini. Scale bar, 15 nm.

FIG. 4 Atomic force microscopy (AFM) images of β-globin pre-mRNA. (a)Unmodified single-stranded RNA; (b) Gold-labeled single-stranded RNA.Black to white spans 6 nm. Scale bar, 100 nm.

FIG. 5 Surface plot of AFM images. The surface plots were drawn from therespective AFM images shown in FIG. 4 using the “surface plot” functionof the NIH Image package. (Left) Unmodified single-stranded RNA(corresponding to FIG. 4 a, left). (Right) Gold-labeled single-strandedRNA (corresponding to FIG. 4 b, right).

DESCRIPTION OF THE PREFERRED EMBODIMENTS

The present invention is of nucleotides comprising a terminal thiolgroup and of nucleotides further comprising a metal cluster, preferablya gold cluster, covalently attached through the terminal thiol group andof nucleic acids comprising at least one, preferably a plurality of thedescribed nucleotides. The present invention can be used in applicationsin which a metal atom is more readily detectable. such as TEM and X-raycrystalography, so as to allow for improved structural analysis ofnucleic acids when complexed with other macromolecules such as proteins.

Methods of labeling oligonucleotides or tRNA with metal atoms are knownin the art, yet these methods are limited to either introduction of suchatoms onto an existing nucleic acid molecule or introduction of suchatoms to a terminal end of an existing nucleic acid molecule.

While conceiving the present invention, it was realized that ifnucleotides to which a metal cluster is covalently linked wereavailable, one could, using conventional template dependent (e.g., DNAand RNA polymerases, reverse transcriptase, etc.) or independentpolymerase (e.g., terminal transferase) based techniques and/orsynthetic solid phase based techniques, to synthesize nucleic acids inwhich the positions of the metal clusters are selected either at random,specific to certain preselected purine or pyrimidine nucleotides, or atknown positions along a nucleic acid molecule.

While reducing the present invention to practice, a model approach ofribonucleotides and RNA labeled with gold clusters was chosen, yet, aswill be appreciated by one of ordinary skills in the art,deoxynucleotides, as well as nucleotide analogs, such as, but notlimited to, dideoxy nucleotides and nucleic acid polymers containingsame can also be prepared and employed as described herein.

As used herein in the specification and in the claims section thatfollows, the terms “nucleotide” or in plural “nucleotides” includenative (naturally occurring) nucleotides, which include a nitrogenousbase selected from the group consisting of adenine, thymidine, cytosine,guanine and uracil, a sugar selected from the group of ribose anddeoxyribose (the combination of the base and the sugar is known asnucleoside), and one to three phosphate groups, and which can formphosphodiester internucleosidyl linkages. However, these terms, as usedherein, further include nucleotide analogs. Such analogs can have asugar analog, a base analog and/or an internucleosidyl linkage analog.In addition, analogs exhibiting non-standard base pairing, such asdescribed in, for example, U.S. Pat. No. 5,432,272, which isincorporated herein by reference, are also included under these terms.Thus, as used herein these terms read on molecules capable of, whileincorporated in a polymer, conventional or unconventional pairing viahydrogen bonding with naturally occurring nucleotides or with nucleotideanalogs exhibiting non standard base pairing and which are present in acomplementary polymer.

As used herein, the term “nucleotide analog” includes nucleotides thatare chemically modified in the natural base (hereinafter “baseanalogs”), in the natural sugar (hereinafter “sugar analogs”), and/or inthe natural phosphodiester or any other internucleosidyl linkage(hereinafter “internucleosidyl linkage analogs”).

Examples of base analogs that can be used according to the inventioninclude, but are not limited to, modified purine and pyrimidine basessuch as, for example, O-methyl, C-methyl, N-methyl, deaza, aza, halo.(F, Br, I), thio, oxo, aminopropenyl, amino, acyl, propynyl, pentynyl,and etheno base derivatives, as well as more drastic modifications suchas replacement of the base by phenyl, and additional analogs asdescribed in Eaton, (1997); Benner et al, (1998); Earnshaw & Gait,(1998) and Sakthivel & Barbas (1998).

Examples of sugar analogs that can be used according to the inventioninclude, but are not limited to, modifications of the β-ribofuranosyland β-2′-deoxyribofuranosyl sugar residues such as, for example,2′-O-methyl, 2′-O-allyl, 2′-O-amino, 2′-deoxy-2′-halo (F, Cl, Br, I),2′-deoxy-2′-thio, 2′-deoxy-2′-amino and dideoxy derivatives, as well ascorresponding α-anomers and hexose analogs, and additional analogs asdescribed in Eaton, (1997); Benner et al., (1998); Earnshaw & Gait,(1998); Groebke et al., (19) and Sakthivel & Barbas (1998).

Examples of internucleosidyl analogs that can be used according to theinvention include, but are not limited to, those in which the naturalphosphodiester linkage is replaced by a linkage such asphosphorothioate, phosphorodithioate, phosphoroamidate,methylphosphonate, and additional analogs as described in Eaton, (1997);Benner et al., (1998); Earnshaw & Gait, (1998) and Sakthivel & Barbas(1998).

Also can be used peptide nucleic acids (PNA), in which the entirephosphate-sugar backbone is replaced with a backbone consisting of(2-aminoethyl) glycine units to which bases are attached throughmethylenecarbonyl bridges and nucleotide analogs which are designed forsolid phase synthesis of oligonucleotides, includingoligodeoxynucleotides and oligodeoxyribonucleotides.

Thus, according to one aspect of the present invention there is provideda nucleotide, as this term is defined above, comprising a terminal thiolgroup at a side chain, which side chain is covalently linked to thepyrimidine or purine base (including analog) of the nucleotide.According to a preferred embodiment of the present invention, the sugarmoiety of the nucleotide is ribose, deoxyribose, dideoxyribose or anyother ribose analog. The nucleotide of the invention may be amonophosphate, a diphosphate, a 3′, 5′-bisphosphate, or a5′-triphosphate. Analogs, as is further detailed above may also be used.

The side chain of the nucleotide of the invention carrying the terminalthiol group may be saturated or unsaturated and has 2-20, preferably2-15, most preferably 2-10, carbon atoms, optionally interrupted byheteroatoms such as O, S or N and/or substituted by groups such as ═O,═NH and/or 1-3 alkyl groups. Examples of the nucleotides of theinvention are shown in Schemes 3-5 hereinbelow.

In another aspect, the invention relates to a nucleotide having a metalcluster covalently linked through a terminal thiol group of a side chaincovalently linked to the pyrimidine or purine base of the nucleotide.The metal may be Ag, Au, Hg, Pt, Mo or W, but is preferably a goldcluster such as colloidal gold.

In a further aspect, the invention relates to a nucleic acid comprisingat least one nucleotide of the invention comprising a free terminalthiol group or a metal cluster covalently linked through a terminalthiol group. The nucleic acid may be a RNA or a DNA molecule.

In one embodiment, the nucleic acid molecule is covalently tagged with ametal cluster. The metal may be Ag, Au, Hg, Pt, Mo or W, but ispreferably a gold cluster such as colloidal gold.

The metal-tagged nucleic acids of the invention are useful as probes formacromolecular assemblies such as protein-RNA complexes.

The invention provides general methodologies for the covalent attachmentof gold-clusters to DNA and RNA (nucleic acids) at random locations aswell as at specific locations.

The general strategy for the attachment of gold-clusters at randomlocations in the nucleic acid molecule is depicted in Scheme 1 andinvolves the following steps:

(i) preparation of precursor deoxyribonucleoside triphosphates (NTPs)and ribonucleoside triphosphates (rNTPs)whose heterocyclic ring containssubstituents with a terminal thiol group (NTP-SH and rNTP-SH,respectively);

(ii) incorporation of these precursor molecules into DNA or RNA inreactions catalyzed by DNA polymerase or RNA polymerase, respectively;and

(iii) attachment of gold-clusters to the free thiol groups, either byreacting with a commercially available maleimido derivative of thecluster, or by reacting with colloidal gold of pre-determined size.

The strategy for the attachment of a gold cluster to a specific locationin the nucleic acid molecule is depicted in Scheme 2 and involves thefollowing steps:

(I) preparation of 3′, 5′ deoxyribonucleoside diphosphates (p[dN]p) and3′, 5′ ribonucleoside diphosphates (pNp) whose heterocyclic ringcontains substituents with a terminal thiol group (p[dN]p-SH and pNp-SH,respectively);

(ii) synthesis of the 5′ half of the nucleic acid whose 3′ endnucleotide is the one that precedes the nucleotide to which a goldcluster should be attached;

(iii) addition of p[dN]p-SH or pNp-SH to the nucleic acid made in (ii)by using the DNA ligase or RNA ligase, respectively; and

(iv) dephosphorylation of the 3′ end of the nucleic acid made in (iii),and ligating it to the 3′ half of the nucleic acid.

The present invention provides the first description of a generalmethodology to covalently label synthetic nucleic acids with metalclusters. The invention is exemplified with respect to RNA labeled withgold clusters. However, in an analogous manner, it may be employed tolabel DNA molecules; namely, by using thiolated dNTP precursors in DNApolymerase driven reactions. The generality of the method is alsomanifested by the possibility it offers to label nucleic acids withclusters of heavy atoms others than gold.

Though colloidal gold was previously used to noncovalently label RNA andprotein RNA complexes, it seems likely that the covalent binding of goldor other heavy metal clusters to biological assemblies of nucleic acidsand proteins is advantageous. First, the binding is stable and direct inthe sense that it does. not require secondary molecules such asantibodies or biotin-avidin complexes. Second, the metal clusters arerelatively small and uniform in size and do not tend to aggregate. Thesefeatures should provide better sensitivity and resolution to the method.Further, the methodology enables the labeling of specific residues alongthe nucleic acid chain (e.g., uridines or adenosines) and also to varythe density of the label by varying the concentration of the thiolatednucleotide during the enzymatically driven polymerization.

The number of studies of biologically important protein-RNA complexes isincreasing rapidly. The extremely large size of many of these complexesmakes the use of microscopic methods essential. The precise location anddefined size of the gold label make this modification suitable forfollowing the RNA path not only within naked RNA, as demonstrated here,but should also allow microscopic visualization of RNA within proteinRNA complexes. For example, visualization of gold-tagged RNA withinunstained frozen hydrated RNP complexes. This methodology is alsoapplicable to help with phase determination in crystals of RNA-proteincomplexes and also in crystals of RNA molecules such as ribozymes.

The general methodology described here is designed to preparegold-tagged RNAs in which gold clusters are randomly distributed alongthe RNA chain. Further developments include the preparation of RNA orDNA molecules tagged with gold clusters at specific locations. Thisallows the localization of specific nucleotides, or sequences ofphysiological importance, within a large variety of protnucleic acidcomplexes, which is a key step toward understanding the mechanism ofaction of such macromolecular assemblies.

The methodology of the present invention enables visualization ofnucleic acids complexed with proteins by electron microscopy as well asuse in microelectronic devices.

Progress in silicon-based microelectronics has led to the shrinkage ofthe characteristic size of a transistor to 0.25 microns in present daystechnology. It is projected that in the course of the next decade thissize will be reduced to ˜0.1 microns. However, major fundamentalphysical considerations make further size reduction very unlikely. It istherefore anticipated that a new and different technology will be neededto reduce the transistor size beyond the 0.1 microns limit.Biological-molecules based technology is a promising candidate for thislength scale.

A possible approach to realize a sub-0.1 microns transistor is thesingle electron transistor (SET). This transistor consists of aconducting small island weakly coupled (by tunneling) to two metalcontacts. The current through the island is of single electrons, whichtunnel in and out of the island. This current is controlled by a metalgate, which can switch in on or off. The required size of the island fora room temperature operation of the SET is ˜10 nm.

The present invention enables forming a structure of a gold cluster at achosen location on a nucleic acid molecule (DNA or RNA). This can be thecritical building block for a SET, with the cluster being the metallicisland. To realize the transistor one has lo construct a structure oftwo contacts with a very small spacing between them, of the order of ˜10nm, and bind the two ends of the nucleic acid molecule to thesecontacts. The length of the molecule (and correspondingly the distancebetween the contacts) defines the tunneling of electrons from the goldcluster to the nearby contacts.

The important properties of the present invention for realizing such adevice are the ability to determine the island size in the run range,the ability to fix its location on the molecule with a very highaccuracy, the ability to determine the molecule length, and the abilityto control the specific location on the molecule, which will bind to themetallic contacts.

Additional objects, advantages, and novel features of the presentinvention will become apparent to one ordinarily skilled in the art uponexamination of the following examples, which are not intended to belimiting. Additionally, each of the various embodiments and aspects ofthe present invention as delineated hereinabove and as claimed in theclaims section below finds experimental support in the followingexamples.

EXAMPLES

Reference is now made to the following examples, which together with theabove descriptions, illustrate the invention in a non limiting fashion.

Example 1 Synthesis of thiolated UTP (Scheme 3)

1a. Synthesis of 5-Aminoallyl-UTP

5-Aminoallyl-UTP was synthesized as described by Langer et al. (Langeret al., 1981). In brief, a mixture of 275 mg of UTP (0.5 mmole, Sigma)and 0.8 gr of mercuric acetate in 50 ml of 0.1 M sodium acetate, pH 6,was stirred at 50° C. for 4 h and cooled on ice. Quantitative formationof the 5-mercurated UTP intermediate was confirmed by TLC analysis on aPEI cellulose plate in 0.75 M KH₂PO₄, pH 3.4 (R_(f) 0.1) where UTPmigrates with an R_(f) value of 0.72. Lithium chloride (196 mg) wasadded to the chilled reaction mixture followed by 6 extractions with 50ml ethyl acetate. The aqueous layer was added to 150 ml of ice coldethanol, the precipitate was collected, washed with ether and driedunder vacuum. The product was resuspended in 25 ml of 0.1 M sodiumacetate, pH 5, and nine-fold molar excess of allyl amine (Merck,neutralized with acetic acid) was added followed by one equivalent ofK₂PdCl₄ (163 mg, Aldrich). The reaction mixture was stirred for 24 h atroom temperature and was then loaded on a column (50 ml bed volume) ofSephadex A-25 equilibrated with 20 mM triethylamoniumbicarbonate, pH 7.8(TEAB). The column was washed with the same buffer and then developedwith a gradient of 0.15-0.8 M TAB. 5-Aminoallyl UTP was eluted at0.3-0.37 M salt. The peak fractions were combined and lyophilized, andthe resulting powder was resuspended in water and re-lyophilized. Thisprocedure was repeated 4-5 times until most of the salt evaporated. Thestructure of the product was confirmed by UV and 2D PMR spectroscopy.

In a similar way, other compounds carrying a radicalCH₂═CH—(CH₂)_(n)—NH₂, wherein n>1, can replace allylamine (n=1).

1b. Synthesis of 5-Thiol-UTP

5-Aminoallyl UTP (0.1 mmole) was dissolved in 8 mH₂O and transferredinto a glass tube containing 2 ml of 1 M triethanol amine, pH 8.4, 0.25M KCl and 25 mM Mg(OAc)₂. A solution of 2-iminothiolane-HCl (274 mg,Sigma) in 0.5 ml of the same buffer was added, the reaction mixture wasleft at 0° C. for 5-15 h and was then loaded on a column (50 ml bedvolume) of Sephadex A-25 equilibrated with 20 mM TEAB. The column waswashed with the same buffer and then developed with a gradient of0.15-0.8 M TEAB. 5-Thiol UTP was eluted at 0.46-0.52 M salt. The peakfractions were combined and lyophilized, and the resulting powder wasresuspended in water and re-lyophilized. This procedure was repeated 4-5times until most of the salt evaporated. The structure of the productwas confirmed by 2D PMR spectroscopy.

Example 2 Synthesis of thiolated ATP (Scheme 4)

2a. Synthesis of N⁶-(carboxymethyl)ATP

The synthesis was adopted from Gebeyehu et al. (Gebeyehu et al., 1987).A mixture of ATP (98.1 mg) and sodium iodoacetate (333 mg) in 3.37 mlH₂O at pH 6.5 was stirred at for 4 days at 30° C. The reaction mixturewas cooled and poured onto 100 ml of chilled ethanol. The precipitatewas collected and dissolved in 12 ml of H₂O. The pH was adjusted to 8.5with 0.1 M NaOH, the solution was heated to 90° C. for 1 h, cooled toroom temperature and loaded on a column of Sephadex A-25 (20 ml bedvolume) which had been equilibrated with 0.1 M TEAB. After washing with0.1 M TEAB, a step gradient from 0.1 to 0.5 M TEAB in 100 ml incrementswas applied. The starting material eluted at 0.4-0.5 M salt.N⁶-(carboxymethyl)ATP was then eluted with 1 M TEAB. The material waslyophilized and analyzed by NMR and TLC.

2b. Synthesis of N⁶-[(6-aminohexyl)carbamoylmethyl]-ATP

N⁶-(carboxymethyl)ATP (15 mg) was dissolved in 0.48 ml of a 1 M aqueoussolution of 1,6 diaminohexane adjusted to pH 4.7 with 5 M HCl.Ethyldimethylaminopropyl carbodiimide (EDC) (5 mg) was added and themixture was stirred for 2 h. Two additional portions of EDC (5 mg each)were added at 30 min intervals. The reaction mixture was cooled on iceand added to 20 ml of a chilled 1:1 mixture of acetone and ethanol. Theprecipitate was collected, dissolved in water and loaded on a column ofSephadex A-25 (20 ml bed volume) which had been equilibrated with 20 mMTEAB. After washing with 20 mM TEAB, a step gradient from 0.1 to 0.5 MTEAB in 100 ml increments was applied. TheN⁶-[(6-aminohexyl)carbamoylmethyl]-ATP was eluted at 0.5 M salt. Theproduct was analyzed by TLC and NMR.

2c. Synthesis of Thiol-ATP

Thiolation of N⁶-[(6-aminohexyl)carbamoylmethyl]-ATP with2-iminothiolane was carried out as described above for 5-thiol-UTP.Thiol-ATP was purified by anion exchange chromatography on Sephadex A-25using a linear gradient of 0.15-0.7 M TEAB. Thio-ATP eluted at 0.5-0.55M salt and recovered by lyophilization.

Example 3 Synthesis of thiol-AMP-PCP

The synthesis of thiol-AMP-PCP (the unhydrolyzable analog of thiol-ATP)was carried out as described above for thiol-ATP starting from AMP-PCP.

Example 4 Synthesis of thiolated 2′(3′),5′-biphosphocytidine (thiol-pCp)(Scheme 5)

4a. Synthesis of pCp

The synthesis of pCp was as described by Hall and Khorana 1955. Inbrief, cytidine (1 gr) was mixed with 5 ml of phosphorylation reagentand incubated at 60° C. in a sealed glass tube for 20 h. Phosphorylationreagent was prepared by dissolving 5 gr of P₂O₅ in 3.75 ml of 85% H₃PO₄.The reaction mixture was diluted with 60 ml of water,the pH was adjustedto 2.1 with 5 M HCl, and boiled for 15 min. The chilled solution wasneutralized (pH 9) with 4.5 M LiOH and loaded onto a column of Dowex 2×8(50 ml bed volume) that had been equilibrated with 20 mM ammoniumbicarbonate (AMBIC). The column was washed with 20 mM AMBIC until allremaining starting material was eluted, and then developed with a lineargradient of 0.1-0.8 M AMBIC. The product pCp eluted at 0.6‥0.7 M AMBICand was recovered by lyophilization.

4b. Synthesis of N⁴-(6-aminohexyl)-pCp

pCp (0.4 gr) was dissolved in 2 ml of H₂O and mixed with 8 ml of a 3.7 Msolution of 1,6 bisaminohexane (pH 7.2). An aqueous solution of Na₂S₂O₅(2.3 gr in 3 ml H₂O) was added to the reaction mixture simultaneouslywith 14 mg of hydroquinone dissolved in a minimum volume of ethanol. Thereaction mixture was stirred at 42° C. for 15 h, cooled on ice, andmixed with 40 ml of ice cold ethanol. The precipitate was collected,dissolved in H₂O and loaded onto a column of Sephadex A-25 (50 ml bedvolume) that had been equilibrated with 20 mM TEAB. The column waswashed with 20 mM TEAB and then developed with a linear gradient of 0.15-0.6 M TEAB. N⁴-(6-aminohexyl)pCp eluted at 0.3-0.35 M salt. Afterlyophilization the product was characterized by PMR and ³¹P-NMR. Theexample given is with 1,6diaminohexane (n=6) but transamination can bedone with any diamine H₂N—(CH2)_(n)—NH₂.

4c. Synthesis of Thiolated pCp

Thiolation of N⁴-(6aminohexyl)-pCp with 2-iminothiolane was carried outas described above for 5-thiol-UTP. The product was purified by anionexchange chromatography on Sephadex A-25 as described above forN⁴-(6-aminohexyl)-pCp. Thiolated pCp eluted at 0.36-0.45 M salt,recovered by lyophilization, and characterized by 2D NMR.

Example 5 Randomly Thiolated RNA

RNA is normally prepared in vitro by transcribing a plasmid containingthe DNA encoding for the desired RNA in the presence of the four rNTPs.Modified RNAs can be prepared by substituting a modified rNTP for all.or for part, of the particular one from which it was derived. Thedensity of modification can be determined by controlling the ratio ofmodified to normal rNTP in the transcription reaction. As a prototypeexample of this strategy we describe below the preparation ofgold-containing β-globin RNA with thiol-UTP as the modified NTP.Analogous procedures will be used to prepare RNA in which gold clustersare attached to adenine or cytidine residues starting from thiol-ATP andthiol-CTP, respectively.

A standard transcription in vitro reaction catalyzed by SP6 RNApolymerase was carried out using the template plasmid pSP64HbΔ6 cut withBamHI, which yields a 497-nt long RNA containing the first two exons andthe intervening intron of the β-globin gene. In addition to ATP, GTP,CTP, and UTP, the reaction mixture contained 10% thiol-UTP(UTP:thiol-UTP =9:1) and ³²P-labeled ATP as a radioactive tracer. TheRNA was recovered by phenol extraction and ethanol precipitation.

Coupling of thiolated RNA to monomaleimido NANOGOLD (Nanoprobe, StonyBrook N.Y.) was carried out according to the manufacturer instructions.Precipitation with ethanol gave an RNA preparation free of the reagent.The RNA was subjected to electrophoresis on an agarose gel andtransferred to a nitrocellulose membrane. The gold-containing RNA wasrevealed by silver enhancement (FIG. 1, lanes 1-3) whereas unmodifiedRNA which had been treated with monomaleimido NANOGOLD did not stain atall (FIG. 1, lane 4). The electrophoretic mobility of the modified RNAcorresponds to an RNA of about 700 nt. It can thus be estimated that themass of the 497-nt β-globin RNA (˜150 kDa) increased by ˜60 kDa. Sincethe molecular mass of NANOGOLD is not known it is not possible toestimate the number of gold clusters per RNA molecule. However, if weassume that NANOGOLD has twice the mass of undecagold (6,000 Da), anestimate of ˜5 clusters per RNA can be made. This is a reasonableestimate, given that the maximum number of thiolated uridines in the RNAis 12 (10% of ˜125) and that the efficiency of incorporation ofthiol-UTP into the RNA is expected to be substantially smaller than thatof UTP.

Preliminary visualization of gold-RNA by bright field transmissionelectron microscopy (TEM) (FIG. 2) showed gold particles arranged atregular distances on a (imaginary) curve, while a control of unmodifiedRNA which had been treated with monomaleimido NANOGOLD gave no signal atall. FIG. 3 is an AFM image showing unmodified RNA (a); gold labeledsingle stranded RNA (b); and gold labeled double stranded RNA (c).

Example 6 Gold-tagged β-globin RNA

The plasmid pSP64HβΔ6, which contains the human β-globin gene (Kraineret al., 1984), was linearized with BamHI and transcribed in vitro toyield a 497-nt transcript comprising the first two exons and the firstintron of β-globin. A standard transcription reaction mixture (in atotal volume of 20 μl) contained 40 mM Tris-HCl, pH 7.9, 10. mM NaCl, 6mM MgCl₂, 20 mM DTT, 2 mM spermidine, 20 units ribonuclease inhibitor(MBI Fermentas), 1 μg linearized DNA, 0.5 mM each of ATP, GTP, UTP, andCTP, and 40 units of SP6 RNA polymerase (MBI Fermentas). For thepreparation of thiolated RNA, UTP-SH or ATP-SH was added to thetranscription reaction mixture (the syntheses of the thiolatednucleoside triphosphates, UTP-SH and ATP-SH, will be publishedelsewhere). Transcription was carried out for 1 h at 37° C., and theresulting 497-nt RNA was recovered by phenol extraction and ethanolprecipitation.

The thiolated RNA was resuspended in 50 μl of 0.1 M sodium phosphate, pH6.4, containing 1 mM EDTA and 2 mM vanadyl ribonucleoside complex(Chirgwin et al., 1979) as an RNase inhibitor. One nanomolemonomaleimido NANOGOLD (Nanoprobes, estimated 10-fold excess) was addedand the reaction mixture was incubated for 6 h at 4° C. The gold-taggedRNA was recovered by two ethanol precipitations.

6a. Transmission Electron Microscopy

RNA was dissolved in water and deposited on ultrathin carbon films thatspanned holey carbon-coated copper grids. Excess solution was blottedwith a wet filter paper and the grids were imaged in a Philips CM-12 TEMoperating at 100 kV.

6b. Atomic Force Microscopy

RNA samples were pipetted onto a chip of freshly cleaved mica andcovered with a second chip that had been washed with pure water (Milli-QPlus system). The RNA suspension was incubated for 5-10 min and then themica sheets were separated and one of them was brought into contact withpure water. Finally, the bulk fluid on the sample was removed quicklywith a stream of wet nitrogen. The samples were probed in air with aNanoscope III AFM instrument (Digital Instruments, Santa Barbara,Calif.) operating in the tapping mode.

Example 7 Concluding Remarks and Discussion

Thus, to verify the feasibility of the approach to covalently tagnucleic acids, RNA in the above Examples, with, for example, goldclusters, whether NANOGOLD can be covalently incorporated into apre-mRNA molecule and then visualized by TEM was put to test. RNA isnormally prepared in vitro by transcribing a plasmid containing the DNAencoding the desired RNA in the presence of RNA polymerase and all fourribonucleoside triphosphates (rNTPs). Modified RNAs can be prepared bysubstituting a modified rNTP for all, or for part, of the particularrNTP from which it was derived. The density of modification can thus bedetermined by choosing the ratio of modified to normal rNTP in thetranscription reaction. Although most commercial RNA polymerasesrecognize modified rNTPs, even with bulky substituents such as biotin(e.g., Langer et al. 1981), without being limited by any theory, it isconceivable that a rNTP attached to a gold cluster with a diameter of1.4 nm would not be as efficiently recognized by the enzyme. Therefore,first, modified rNTP containing free thiol groups were incorporated intothe RNA and then it was coupled to a maleimido-gold cluster(monomaleimido NANOGOLD). This general strategy is schematicallydepicted in FIG. 1, and as a prototype example we describe below thepreparation and visualization by TEM and Aof gold-tagged β-globin RNA.

Thiolated β-Globin RNA

The plasmid pSP64HβΔ6, which contains the human β-globin gene (Kraineret al, 1984), was linearized with BamHI and transcribed in vitro withSP6 RNA polymerase to yield a β-globin pre-mRNA like molecule harboringthe β-globin first two exons and the intervening intron. To preparethiolated β-globin RNA, the linearized β-globin plasmid was transcribedin vitro with SP6 RNA polymerase in the presence of all four rNTPs and aUTP analog containing a substituent with a terminal thiol group on itsheterocyclic ring (UTP-SH; 5, 10, or 50% of total input UTP). To verifythat UTP-SH was indeed incorporated into the RNA, we performed inparallel a second identical transcription reaction except that[α-³²P]ATP was added as a radioactive tracer. The labeled RNA wasrecovered by phenol extraction and ethanol precipitation and coupled tobiotin using 1-biotin-4-[4′-maleimidomethyl)cyclohexanecarboxyamido]butane (Biotin-BMCC,Pierce), and the resulting RNA was bound to immobilized monomeric avidin(Pierce). Elution of the bound material with D-biotin showed that 85% ofthe input ³²P-labeled RNA was specifically and reversibly retained onthe solid matrix, indicating that the transcribed RNA containedthiolated nucleotides.

A similar protocol was used to prepare β-globin RNA with thiolatedadenosine residues. In that case, the transcription reaction was carriedout in the presence of all four rNTPs and an ATP analog containing asubstituent with a terminal thiol group on its heterocyclic ring(ATP-SH; 10 or 50% of total input ATP).

Gold-Tagged β-Globin RNA

Coupling of thiolated (UTP-SH) β-globin RNA to monomaleimido NANOGOLDwas carried out according to the manufacturer's instructions.Precipitation with ethanol gave an RNA preparation free of the reagent.To demonstrate that NANOGOLD was covalently bound to the RNA, the RNAwas subjected to electrophoresis on an agarose gel and transferred to anitrocellulose membrane. The gold-containing RNA was revealed by silverenhancement (Burry et al., 1992) (FIG. 2, lanes 1-3), whereas unmodifiedRNA that had been treated with monomaleimido NANOGOLD did not stain atall (FIG. 2. lane 4). The apparent electrophoretic mobility of themodified RNA corresponds to RNA of about 700 nucleotides (nt). It canthus be estimated that the mass of the 497-nt β-globin RNA (˜150 kDa)increased by ˜60 kDa. Since the molecular mass of NANOGOLD is not known,it is not possible to estimate the number of gold clusters per RNAmolecule. However, if we assume that NANOGOLD has twice the mass ofundecagold (6000 Da), an estimate of ˜5 clusters per RNA can be made.This is a reasonable estimate, given that the maximum number ofthiolated uridines in the RNA is 12 (10% of ˜125) and that theefficiency of incorporation of thiol-UTP into the RNA is expected to besubstantially smaller than that of UTP.

Visualization of Gold-Tagged β-Globin RNA by TEM

Visualization of unstained gold-tagged β-globin RNA by bright field TEMshowed spots of gold clusters. with a diameter of 1-2 nm, arranged atnearly regular distances on an imaginary curve (FIGS. 3 b and 3 c). Theapparent variability in the size of these spots is similar to thatobserved in TEM images of NANOGOLD and may thus reflect size variationsin a particular batch of the reagent. Though the organic shell ofNANOGOLD is expected to completely eliminate aggregation of theseclusters (Hainfeld and Furuya, 1992), the extremely large spot in FIG. 3b may be attributed to an aggregate that could have been formed duringsample manipulations. Notably, the density of gold clusters along thesecurves corresponds to the proportion of NTP-SH that was present in thetranscription reaction. Thus, gold clusters in β-globin RNA that wastranscribed in the presence of 10% ATP-SH (10% of total input ATP; FIG.3 b) are more spread than those in β-globin RNA that was transcribed inthe presence of 50% UTP-SH (50% of total input UTP; FIG. 3 c). In acontrol experiment, where unmodified RNA had been treated withmonomaleimido NANOGOLD and then manipulated in exactly the same manneras in experiments with thiolated RNA, a signal of scattered goldclusters could only rarely be observed by bright-field TEM (FIG. 3 a).

Visualization of Gold-Tagged β-Globin RNA by AFM

Direct evidence for the attachment of gold clusters at random locationson the RNA was achieved by the simultaneous visualization of RNA andgold clusters by AFM imaging. Diluted RNA samples were deposited onfreshly cleaved mica chips, dried in air, and imaged in air (FIG. 4).The AFM images of single-stranded β-globin RNA (FIG. 4) correspond to anapparent height of about 0.7 run. This value is significantly smallerthan 1.6 nm which is the expected diameter based on X-ray crystalstructure data for the single-stranded helical form of poly(A) (Saenger,1984a,b). On the other hand, it is similar to the height of 0.7±0.1 nmthat was observed by AFM for poly(A) RNA (Smith et al., 1997),indicating that compression of the RNA occurred upon depositing anddrying on the mica. The end to end length in each of the RNA images wasestimated from measurements of their contour length using the NIH Imagepackage and was found to range between 350 and 400 nm. For a 497-nt longRNA, this value corresponds to a nucleotide to nucleotide distance of0.7-0.8 nm, more than two times larger than the 0.282 nm axial rise pernucleotide in single-stranded helical poly(A) RNA (Saenger, 1984b). Thisobservation indicates that stretching of the helical RNA moleculesoccurred during the sample preparation for the AFM and is consistentwith the observed reduction in their diameter.

Notwithstanding, single-stranded gold-tagged β-globin RNA moleculesexhibit images in which knob-like structures are clearly seen along theRNA chai (FIG. 4 b). The height of the brightest knobs (typical knobsare indicated by arrows in FIG. 4 b) range between 1.7 and 2.5 nm. Thisvalue is in agreement with the height observed for free NANOGOLD(2.0-2.6 nm). The distinction between the apparent uniform height ofunmodified RNA and the spiky appearance of the gold-labeled RNA canclearly be seen in the surface plot of the respective RNAs (FIG. 5).These knobs are thus attributed to gold clusters attached to the RNAchain.

Although the invention has been described in conjunction with specificembodiments thereof, it is evident that many alternatives, modificationsand variations will be apparent to those skilled in the art.Accordingly, it is intended to embrace all such alternatives,modifications and variations that fall within the spirit and broad scopeof the appended claims. All publications, patents and patentapplications mentioned in this specification are herein incorporated intheir entirety by reference into the specification, to the same extentas if each individual publication, patent or patent application wasspecifically and individually indicated to be incorporated herein byreference. In addition, citation or identification of any reference inthis application shall not be construed as an admission that suchreference is available as prior art to the present invention.

References

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1. A nucleotide comprising a sugar moiety selected from a natural sugarmoiety and a sugar analog thereof, a natural phosphodiester or any otherinternucleosidyl linkage, and a natural pyrimidine or purine base or abase analog thereof, and a terminal thiol group at a side chain beingcovalently linked to the pyrimidine or purine base or base analog of thenucleotide, said side chain having at least 7 carbon atoms, isinterrupted by at least one heteroatom selected from the groupconsisting of O, S and N and is substituted by at least one ═NH group.2. The nucleotide of claim 1, comprising a natural sugar moiety, anatural phosphodiester linkage, and a natural pyrimidine or purine base,and a terminal thiol group at a side chain being covalently linked tothe pyrimidine or purine base of the nucleotide.
 3. The nucleotide ofclaim 1, wherein the sugar moiety is ribose.
 4. The nucleotide of claim1, wherein the sugar moiety is deoxyribose.
 5. The nucleotide of claim1, wherein the sugar moiety is dideoxyribose.
 6. The nucleotide of claim1, which is a monophosphate, diphosphate, 3′,5′-bisphosphate or5′-triphosphate.
 7. The nucleotide of claim 1, wherein said side chainis saturated or unsaturated and has 7-20 carbon atoms.
 8. the nucleotideof claim 7, wherein said saturated or unsaturated side chain has 7-15carbon atoms.
 9. The nucleotide of claim 1, further comprising a metalcluster being covalently linked through said terminal thiol group atsaid side chain to the pyrimidine or purine base of the nucleotide. 10.The nucleotide of claim 9, wherein said metal is Ag, Au, Hg, Pt, Mo orW.
 11. The nucleotide of claim 10, wherein said metal is Au.
 12. Thenucleotide of claim 11, wherein said metal cluster is colloidal gold.13. A nucleic acid comprising at least one nucleotide of claim
 1. 14.The nucleic acid of claim 13, comprising ribonucleotides.
 15. Thenucleic acid of claim 13, comprising deoxyribonucleotides.
 16. Thenucleic acid of claim 13, wherein said at least one nucleotide furtherincludes a metal cluster covalently linked through said terminal thiolgroup at said side chain to the pyrimidine or purine base or base analogof the nucleotide.
 17. The nucleic acid of claim 16, wherein said metalis Ag, Au, Hg, Pt, Mo or W.
 18. The nucleic acid of claim 17, whereinsaid metal is Au.
 19. The nucleic acid of claim 18, wherein said metalcluster is colloidal gold.
 20. A method for labeling a nucleic acidmolecule at random locations with a metal, the method comprisingincorporating a thiolated nucleotide according to claim 1 into saidnucleic acid molecule, and attaching the metal atoms to the free thiolgroups of the thiolated nucleic acid.
 21. The method of claim 20 for theattachment of gold-clusters at random locations in a nucleic acidmolecule, comprising: (i) preparation of precursor deoxyribonucleosidetriphosphates (NTPs) and ribonucleoside triphosphates (rNTPs) whoseheterocyclic ring contains substituents with a terminal thiol group(NTP-SH and rNTP-SH, respectively); (ii) incorporation of theseprecursor molecules into DNA or RNA in reactions catalyzed by DNApolymerase or RNA polymerase, respectively; and (iii) attachment ofgold-clusters to the free thiol groups, either by reacting with acommercially available maleimido derivative of the cluster, or byreacting with colloidal gold of pre-determined size.